The invention relates to methods for the identification of compounds that modulate the activity of target proteins having motor domains and use of such methods for the identification of therapeutic agents.
The kinesin superfamily is an extended family of related microtubule motor proteins. It can be classified into at least 8 subfamilies based on primary amino acid sequence, domain structure, velocity of movement, and cellular function. This family is exemplified by xe2x80x9ctruexe2x80x9d kinesin, which was first isolated from the axoplasm of squid, where it is believed to play a role in anterograde axonal transport of vesicles and organelles (see, e.g., Goldstein, Annu. Rev. Genet. 27:319-351 (1993)). Kinesin uses ATP to generate force and directional movement associated with microtubules (from the minus to the plus end of the microtubule, hence it is a xe2x80x9cplus-end directedxe2x80x9d motor).
KIF (KInesin Family) proteins are microtubule-dependent molecular motors that play important roles in intracellular transport and cell division. More specifically, several of these kinesins have been found associated with the arms of mitotic chromosomes. These kinesins are thought to provide a force, sometimes referred to as the polar ejection force, that is directed away from each spindle pole and is thought to contribute to movement of mitotic chromosomes toward the mitotic spindle midzone during prometaphase of mitosis. These kinesins are sometimes collectively referred to as chromokinesins.
Within this functional group of kinesins resides a group of kinesins from several organisms that share significant sequence homology. These include human Kif4 (HsKif4), murine Kif4 (MmKif4), and Xenopus laevis XKlp1, which are all closely related and are probably functional orthologs. Drosophila nod, and Drosophila Klp3A and C. elegans ChromoK-A and -B are more distantly related, but also appear to function during mitosis.
No studies of human Kif4 have been reported in the literature. cDNA sequence spanning the coding region of the HsKif4 RNA is entered under two Genbank accesssion numbers, AF071592 and AF179308.
A full length version of Kif4b, the Chromosome 5 Kif4 variant, has been identified. The two proteins are 93.9% identical overall, 97.1% in the motor domain. The DNA sequences share 95% identity in a 3845 bp overlap which includes some non-coding sequence.
The murine kinesin MmKif4 was identified in a PCR-based search for novel kinesins in the mouse nervous system, and appears as full length sequence in Genbank under the Accession number NMxe2x80x94008446. This gene is expressed in several tissues during early development, but is largely absent from adult tissues, with the exception of the spleen. Full length cloning of the MmKif4 cDNA; expression of full length MmKif4 in insect cells using a baculovirus expression vector; measurement of microtubule-stimulated ATPase of MmKif4; and analysis of MmKif4 microtubule binding and gliding activities have also been reported. MmKif4 has also been reported to interact with the murine leukemia virus Gag polyprotein.
XKlp1 is the Xenopus laevis ortholog of MmKif4. The full length sequence of XKlp1 has been reported. See, Accession number X82012. Xklp1 expression was found to be most prominent in gametes and dividing cells, and was localized to interphase nuclei, mitotic chromosomes, and to the spindle midzone. Perturbation of XKlp1 function by either antisense oligonucleotide injection into developing Xenopus embryos, or by antibody addition to mitotic spindle assembly reactions in vitro led to dramatic defects in mitotic spindle formation.
Chromokinesin was identified as an mRNA expressed in early embryonic neurons of the chicken. The initial cDNA clone did not include the motor domain, and chromokinesin was not identified as a kinesin until the remainder of the cDNA clone was identified. Chicken chromokinesin was found to be expressed preferentially, if not exclusively in proliferating cells of the developing chick retina, and was localized to the nuclei of interphase cells, and to chromosomes of mitotic cells.
Defects in function of these proteins would be expected to cause a failure in prometaphase chromosome alignment resulting in cell cycle arrest in mitosis. As such, compounds that modulate the activity of the chromokinesins may affect cellular proliferation. The present invention provides a novel method to identify such compounds.
The present invention provides methods to identify candidate agents that bind to a target protein or act as a modulator of the binding characteristics or biological activity of a target protein. In one embodiment, the method is performed in plurality simultaneously. For example, the method can be performed at the same time on multiple assay mixtures in a multi-well screening plate. Furthermore, in a preferred embodiment, fluorescence or absorbance readouts are utilized to determine activity. Thus, in one aspect, the invention provides a high throughput screening system for detecting modulators of activity a target protein.
In one embodiment, the present invention provides a method of identifying a candidate agent as a modulator of the activity of a target protein. The method comprises adding a candidate agent to a mixture comprising a target protein which directly or indirectly produces ADP or phosphate, under conditions that normally allow the production of ADP or phosphate. The method further comprises subjecting the mixture to a reaction that uses said ADP or phosphate as a substrate under conditions that normally allow the ADP or phosphate to be utilized and determining the level of activity of the reaction as a measure of the concentration of ADP or phosphate. A change in the level between the presence and absence of the candidate agent indicates a modulator of the target protein.
The phrase xe2x80x9cuse ADP or phosphatexe2x80x9d means that the ADP or phosphate are directly acted upon by detection reagents. In one case, the ADP, for example, can be hydrolyzed or can be phosphorylated. As another example, the phosphate can be added to another compound. As used herein, in each of these cases, ADP or phosphate is acting as a substrate.
Preferably, the target protein either directly or indirectly produces ADP or phosphate and comprises a motor domain. More preferably, the target protein comprises HsKif4, HsKif4b, MmKif4, XKlp1, Drosophila nod, Drosophila Klp3A, or C. elegans ChromoK-A or -B, or a fragment thereof.
Also provided are modulators of the target protein including agents for the treatment of cellular proliferation, including cancer, hyperplasias, restenosis, cardiac hypertrophy, immune disorders and inflammation. The agents and compositions provided herein can be used in variety of applications which include the formulation of sprays, powders, and other compositions. Also provided herein are methods of treating cellular proliferation disorders such as cancer, hyperplasias, restenosis, cardiac hypertrophy, immune disorders and inflammation, for treating disorders associated with HsKif4 activity, and for inhibiting HsKif4.